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Expression

By Debbie Dunn,2014-07-23 14:31
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ExpressionExpres

    Expression

May2008,Volume2,No.5(SerialNo.6)JournalofLifeSciences,ISSN1934-7391,USA

    ;Expressionofthechemoresistancerelatedproteinsandp53geneasa

    ;predictorofchemotherapyresponseandaprognosticfactorin ;patientswithovariancanceratthestagesofIIIV

    ;MAJia-jia,CHENBiliang,XINXiao-yan

    ;(DepartmentofObstetricandGynecology,XijingHospital,Fou~hMilitaryMedicalUniv

    ersity,Xi’an710033,China

    ;Abstract:ObjecfiveToclarifytheprognosticsignificanceof ;histologicsubtypeanditscorrelationtoexpressionof ;chemoresistance-relatedproteins(CRPs)inovariancancer. ;MethodsAtotalof107stageII.IVovariancancers.wherethe ;proportionofserous,endometrioid,mucinous,andclearcell ;subtypewas62.6%,15.9%,14.0%,and7.5%,respectively,were ;investigatedforglutathioneStransferasepi(GST-pi),MDR

    ;(multidrugresistance)-1,andp53expressionusing ;immunohistochemistry.ResultsGST-piexpressionwas ;detectedin62.6%ofthetumorsandwasnotrelatedtohistologic ;subtypeoftumor.MDR1expressionwasobservedin52.3%of

    ;thetumorstestedandwasmorefrequentlydetectedinserous ;adenoearcinomasthanotherhistologicsubtypesoftumor ;(p(0.0o1).P53expressionwasfoundin43.3%ofserous,35.3% ;ofendometrioid.40.0%ofmucinousadenocarcinomasand ;37.5%ofcleareelladenocarcinomas.Inunivariateanalysis. ;therearedirecteorrelationsbetweenCRPsandoverallsurviva1. ;Inmultivariateanalysis,GST-piexpression(P--0.0052),MDR1

    ;expression(P=0.0058),histologicsubtype(P=O.0067),FIGO ;stage(P-0.0089),andresidualtumor(P=O.0041)werefoundto ;besignificantindependentprognosticfactors.Conclusion ;Histologicsubtypeprovedtobethesignificantindependent ;prognosticfactorinadditiontoF1GOstageandresidualtumorin ;stageIIIVovariancancer.GST-pi.MDR1.andp53expression

    ;patterniscloselyrelatedtohistologicsubtypeofovariancancer, ;atthesametimetheyarethesignificantpredictorsofsurviva1. ;Keywords:chemoresistancerelatedprotein;GST-pi;MDR1:

    ;ovariancancer;p53

    ;1.Introduction

    ;Cancerchemotherapyhasgraduallyimproved

    ;withthedevelopmentofnovelantitumordrugsandhas ;profound,positiveresultswhenappliedtomany

;MAJia-jia(1981),female,Ph.D.;researchfield

    ;gynecologyoncology.

    ;10

    ;hematologicmalignancies,andsomesolidtumors, ;especiallygermcellandsomechildhood

    ;malignanciesl_2.Whiletreatmentofcertain

    ;malignancieswithchemotherapyhasbeensuccessful ;andencouraging,theeffectivenesshasoftenbeen ;limitedbydrugresistanceoftumorsandbysideeffects ;onnormaltissuesandcells.Infact,manytumorsare ;intrinsicallyresistanttomanyofthemorepotent ;cytotoxicagentsusedincancertherapy.Othertumors, ;initiallysensitive,recurandareresistantnotonlytothe ;initialtherapeuticagents,butalsotootherdrugsnot ;usedinthetreatment.

    ;Surgeryandchemotherapyarethemaintreatment ;modalitiesforovariancancel’.Oneofthemost

    ;importantprognosticfactorsforovariancanceris ;tumorchemoresistance3_4.Treatmentfailureof

    ;ovariancancerismostlyrelatedtoresistanceto ;chemotherapythatthetumorsacquireduringtreatment ;orthatiSintrinsictothetumors.Becauseoftheserious ;problemofclinicaldrugresistance,muchefforthas ;beenexpendedtoadvanceourunderstandingofthe ;mechanismsofdrugresistanceincancercells.The ;molecularmechanismsofdrugresistance,however,are ;multifactorial,andincludedrugtransportmechanisms, ;DNArepairsystems,detoxifyingenzymes,and ;apoptosisinhibition.

    ;Nowadays,alargenumberofcytotoxicagents, ;includingplatinumcompounds,taxanes,

    ;topoisomeraseIandIIinhibitors,andantimetabolites ;havebeenusedinthetreatmentofadvancedovarian ;Expressionofthechemoresistance-relatedproteinsandp53geneasapredictorofchemothe

    rapyresponse

    ;andaprognosticfactorinpatientswithovariancanceratthestagesofII?IV——

    ;cancers.Failureofthetumortorespondtosuchagents ;ismediatedinsignificantpartbythephenomenonof ;multidrugresistance(MDR),wherebycytotoxicagents ;areactivelyexcludedfromcompartmentsofthe ;intracellularenvironment.Transmembraneprotein ;(Pglycoprotein,Pgp)intracellularproteins

    ;(glutathioneStransferases,GSTs)andtumor

    ;suppressorgenep53havebeenimplicatedinthis

    ;phenomenon.whichisreversibleinmanyinvitro ;cancermodelsystems.However.ithasnotyetbeen ;clarifiedwhethertheexpressionprofileof ;chemoresistancerelatedproteinsfCRPs)isrelatedto ;thespecificsubtypeofovariancarcinomaand ;predictiveforchemoresponsivenessofthetumor. ;Theaimofthepresentstudywastoevaluatethe ;correlationofMDR1,GSTpi,andp53expression

    ;withhistologicsubtypeofovariancarcinomaandtheir ;prognosticsignificanceinaretrospectiveseriesof93 ;stageII.IVovariancancers.

    ;2.Materialandmethods

    ;2.1Patients

    ;TheI1IGOstageofthepatientswasasfollows:18 ;stageII,68stageIII,and21stage1V.Ageofpatients ;rangedfrom36to79(median:58)years.Patientswere ;operativelytreatedattheDepartmentofObstetricsand ;Gynecology,Xijinghospital,theaffiliatedhospitalsof ;FourthMilitaryMedicalUniversitybetweenJanuary ;2000andAugust2003.Althoughtheperiodofpatient ;entryisrelativelylong,thetreatmentstrategywas ;uniform.Histologicsubtypewasdeterminedbyusing ;thecriteriaforthediagnosisaccordingtoFIGOandthe ;Wor1dHealthOrganization.Distributionoftumor ;subtypewasasfollows:67serous(62.6%),17 ;endometrioid(15.9%),15mucinous(14.0%),and8 ;cleareel1adenocarcinomasr7.5%1.Undifierentiated ;carcinomas,transitionaleellcarcinomas,and ;squamouscellcarcinomaswereexcludedfromthe ;study.Gradeoftumorwasdeterminedaccordingto ;architecturalpattern,nucleargrade,andmitosisindex. ;Noneofthepatientsreceivedchemotherapypriorto ;firstlaparotomy.

    ;2.2Surgicaltreatmentandchemotherapy

    ;Inourtreatmentstrategyforovariancancer,we ;performeddebulkingsurgerytoobtainoptimal ;cytoreduction(residualtumor<2cm)atfirst ;laparotomy.Surgicalprocedureincludedextended ;totalhysterectomy,bilateralsalpingooophorectomy,

    ;omentectomy,andresectionofmetastatictumors. ;Systematicpelvicandparaaorticlymphnode

    ;dissectionwasemployedwhentheoptimal

    ;cytoreductionwasachieved.Patientswhoobtained ;optimalstatusreceivedsixcyclesofplatinumbased

    ;chemotherapy.Patientswitharesidualtumor_>2cm ;receivedtwotothreecyclesofchemotherapyand ;underwentsecondarycytoreductivesurgery,including ;systematiclymphadenectomy,unlessdiseasewas ;consideredprogressive.Althoughweregardedpatients ;havebeenoptimallycytoreducedwhentheresidual ;tumorbecame<2cmateitherfirstsurgeryor ;secondarycytoreductivesurgery,residualtumoratfirst ;surgerywasemployedasaprognosticvariableinthis ;study.Thefirstlinechemotherapyconsistedof

    ;cisplatin(5070mm),adriamycin(40mg/m),and

    ;cyclophosphamide(350mg/m’)every21days,

    ;paclitaxel(175mg/m)andcarboplatin(AUC~5) ;combinationevery21daysaschemotherapy.

    ;2.3Tumortissuesandimmunohistochemistry ;Allthetumorsampleswereobtainedatfirst

    ;laparotomy.Tumortissueswerefixedin10%formalin ;andembeddedinparaffinand4mmserialsections

    ;wereimmunostainedforGST-pi,MDR1,andp53

    ;usingthestreptavidin--biotin--peroxidasecomplex ;method.Theslideswereincubatedfor1hat110.C, ;dewaxedinthreexylenebathsfor1mineach,and ;rehydratedinanalcoholseries(100%,85%,7O%).The ;antigenretrievalstepwasperformedbyincubatingthe ;slidesinCitrateBuffer(pH6.01for2microwave ;cyclesof7minat700W.Afterbeencooleddownat ;roomtemperaturefor15min,theperoxidaseactivity ;wasblockedwith3%hydrogenperoxideinmethanol ;11

    ;Expressionaftheehemoresistance.relatedproteinsandp53geneasapredictorofchemothe

    rapyresponse

    ;andaprognosticfactorinpatientswithovariancanceratthestagesof?-IV

    ;for15min.andtheslideswererinsedindistilledwater. ;Theslideswereincubatedwiththeprimaryspecific ;antibodyagainstGSTpi(Sigma),MDR1(Sigma),

    ;andp53(Sigma)for30minat37.Cinamoisturizing ;chamber.AfterrinsingwithPBSbuffer(pH7.6,at ;roomtemperature,theslideswereincubatedwiththe ;secondaryantibodyfor1hat37.C,followedbythree ;rinsesof5mineachinPBS.Theslideswereincubated ;for20minat37.Cwithstreptavidinperoxidaseand

    ;washedindistilledwater.Reactionwasvisualized ;using3,30diaminobenzidineaschromogen.The

    ;slideswerecounterstainedwithMayer’shematoxylin

    ;forlmin,dehydratedinalcoholseries,mountedin ;EntellanMountingMedia(Sigma),andcoveredwith ;glasscoverslip.Tissuespecimensoftheuterine ;papillaryserousadenocarcinomaknownforpositive

    piandp53wereusedforpositive ;expressionforGST

    ;controlandepitheliaofthelargeintestinewereusedfor ;positivecontrolforMDR1.

    ;2.4Evaluationofimmunostaining

    ;Anaverageof300cellswasscoredineachof

    ;threeseparatetumorareas.ForGSTpi,onlythecells

    ;withbrowncoloredcytoplasmicstainingwas

    ;consideredpositiveexpression.ForMDR1andp53,

    ;onlystainingoncellmembraneandnuclearstaining ;wereconsideredpositiveexpression,respectively. ;Backgroundandcytoplasmicstainingwerenotcounted ;inanyofthecases.Fromtheproportionofpositive ;cellsandtheimmunoreactivityforGSTpiandMDR1,

    ;theauthorsclassifiedtumorsintotwocategoriesfor ;eachproteinasfollows:negative6-)when010%of

    ;thecellsstainedstronglyintheirnucleus;andpositive ;whenover10%ofthecellsstainedpositive.Asforp53. ;theresultswereclassifiedas:negative(_)whenless ;than5%ofthecellsstainedstronglyintheirnucleusor ;membrane;positive()when5-50%ofthecells

    ;stainedstronglyintheirnucleusormembrane;and ;strongpositive(++)whenmorethan51%ofthecells ;stainedstronglyintheirnucleusormembrane. ;2.5Statisticalanalysis

    ;l2

    ;Correlationbetweenthevariableswasanalyzed ;usingxz—testorFisher’sexacttest.Overal1survival

    ;curveswerecomparedusingKaplanMeiermethodand

    ;logranktest.Univariateandmultivariatesurvival ;analyseswereperformedusingCoxregressionmodel ;withoverallsurvival(oS)astheoutcomemeasure. ;Forwardstepwiseprocedurewasusedtoselectthe ;independentvariableinmultivariateanalysis.P.values ;lessthan0.05wereregardedassignificant. ;3.Results

    ;0f107ovariancarcinomas,67(62.6%)were

    ;positiveforGSTpi,56(52.3%)werepositivefor

    ;MDRl,and44(41.1%)werepositiveforp53

    ;expression.TherelationbetweenGSTpi,MDRl,and

    ;p53expressionandtheclinicopathologic

    ;characteristicsofpatientsareshowninTable1.We ;foundanegativecorrelationbetweenMDR-1andp53 ;expressionrjP>0.05).

    ;Table1CorrelationofGST-pi,MDR-1andp53

    expressionwithclinicopathologicalfeatures ;

    ;ofpatientswithstagesII-IV

    ;Expressionofthechemoresistance-relatedproteinsandp53geneasapredictorofchemothe

    rapyresponse

    ;andaprognosticfactorinpatientswithova~ancanceratthestagesofH-IV

    ;TheproportionofMDR1positivetumorswas

    ;significantlydifferentamongthehistologicsubtypesof ;tumor.Only2of15(13.3%)mucinous

    ;adenocarcinomasandoneof8(12.5%)clearcell ;adenocarcinomashadMDR1expression.Sixof17

    ;(35.3%,endometrioidadenocarcinomasshowed ;positiveMDR1staining.Incontrast,47of67(70.1%) ;serousadenocarcinomashowedpositiveMDR1

    ;staining(Fig.1A,1B).Theproportionoftumorswith ;positiveMDR1expressioninserousadenocarcinoma ;wassignificantlyhigherthanthatinotherhistologic ;subtypesoftumorfP<0.05).

    ;A

    ;C

    ;B

    ;E

    ;D

    ;F

    ;Fig.1Representativeimmunohistochemicalstainingforchemoresistance-relatedprotein

    s

    ;Notes:fA)PositivestainingforMDR

    1inaserousadenocarcinoma(×200);(B)PositivestainingforMDR1inaendometrioid ;adenocarcinoma(×200);(C)PositivestainingforGST-piinaserousadenocarcinoma(×20

    0);(D)Positiveimmunostainingfor

    ;GST-piinacaseofclearcellcarcinoma(×200);(E)1+immunostainingforp53inacaseofser

    ousadenocarcinoma(x200);(F)2+

    ;immunostainingforp53inacaseofendometrioidadenocarcinomaf×200).

    ;GSTpiexpressionhadnorelationshipwith

    ;histologicsubtypeoftumor.PositiveGSTpistaining

    ;(Fig.1C,1D)wasobservedin43/67(64-2%)ofserous, ;10/17(58.8%)ofendometrioid,9/15(60.0%)of ;mucinous,and5/8(62.5%)ofclearcell

    ;adenocarcinomas.P53expressionasclassifiedinto1+ ;and2+(Fig.1F,1E)inserous,endometrioid,mucinous ;andclearcelltumorswas29/67(43-3%),6/17(35.3%),

    ;6/15(40.0%)and3/8(37.5%),respectively. ;13

    ;Expressionofthechemoresistanee?relatedproteinsandp53geneasapredictorofchemoth

    erapyresponse

     ;andaprognosticfactorinpatientswithovariancanceratthestagesofH-IV————

    ;Inunivariatesurvivalanalysis,GSTpiand

    ;MDR?1wasfoundtohaveasignificantcorrelation ;withoverallsurvival(os)(P<0.05,Fig.2A,2B).We ;foundnoprognosticsignificanceintheexpressionof ;p53,ageofthepatientsandtumorgrade.Histologic ;subtypewasrelatedtoOS(P<0.05,Fig.3).Inthe ;analysis,histologicsubtypewascategorizedinto ;serous/endometrioidtumorsandmucinous/clearcell ;tumorsbasedontheestimated3-yearsurvivalfor ;serous,endometrioid,mucinous,andcleareelltumors ;being50.4%,48.8%,34.3%,and35.6%,respectively. ;AsforFIGOstage(?VS.Ill/IV,Fig.4)andresidual

    ;tumor(<2cmvs->2cm,Fig.5),thesefactorswere ;significantlycorrelatedwithOS(P<0.05)(Table2).In ;multivariateanalysis,GSTpiexpression(P=0.0052),

    ;MDR-1expression(P0.0058),histologicsubtype ;(P:O.OO67),HGOstage(P=0.0089),andresidual ;tumorrP=0.0041)werefoundtobesignificant ;independentprognosticfactors(Table2). ;14

    ;OO0

    ;尊蜘棚嘲嘲

    ;A

    ;l

    ;JnIlime(mol1sI

    ;B

    ;Fig.2Overallsurvivalprobabilityinrelationto ;chemoresistance-relatedproteins

    ;Notes:A,MDR1;B,GST-pi.

    ;Fig.3Overallsurvivalprobabilityinrelation ;tohistoloatedproteinsandp53geneasapredictorofchemotherapyresponse

    ;andaprognosticfactorinpatientswit

    ;hovari!!!!!!g!!:

    ;Table2UnivariateandmultivariateCOXregression ;analysesforprognosticfactorsofstages

    ;?.IV0vaancancercases

    ;Notes:HR:hazardratio;CI:confidenceinterval;Ref: ;referenceforhazardratio.

    ;4.Discussion

;TheGSTsareafamilyofenzymesthatare

    ;responsiblefortheconjugationofsome

    ;chemotherapeuticagentswithglutathioneandare ;implicatedintheresistancetoanumberofdrugs.There ;havebeenconflictingreportswhetheranalysisfor ;GST-picanpredicttheresponsetochemotherapyof ;ovariancancerSomestudiesfailedtorevealthe ;relationshipbetweenGSTpiexpressionandresponse

    ;tochemotherapyorsurvival,whereasotherauthors ;foundpositiverelationbetweenGSTpiand

    ;chemotherapyresponseinovariancancer.Mdr1gene

    ;encodesP170(PgP),whichisatransmembrane

    ;transportproteinandfunctionsasanenergydependent

    ;effiuxpumpforanumberofdrugsandisresponsible ;fordecreaseddrugaccumulationwithincells.MDR1

    ;proteinisthoughttobeapredictorofresponseto ;chemotherapyinovariancancer.Thep53tumor ;suppressorgeneencodesanuclearphosphoprotein ;involvedincellcyclecontrol,DNArepair,and ;apoptosis.InresponsetoDNAdamage.Becausemany ;ofthemissensemutationsofp53proteinareresistantto ;degradationandmorestable,theycanbedetectedin ;tumortissuesbyimmunohistochemicaltechniques. ;P53alterationshavebeensuggestedtoberelatedto ;resistancetocisplatinbasedchemotherapyandmore

    ;aggressivebehaviorofovariancancersI67..

    ;Althoughplatinumbasedchemotherapy

    ;increasedtheresponserateofovariancancers,tumors ;candevelopchemoresistanceandsometumorshave ;intrinsicchemoresistance,amaincauseoftreatment ;failureofthisdisease.Chemoresistanceofmalignant ;tumorsisrelatedtomultipleclassesofCRPs,which ;includemembrane-boundtransportermolecules, ;detoxifyingenzymes,DNArepairsystems,and ;aberrationinapoptosismechanisms.Ithasnotbeen ;clarifiedwhethertheexpressionprofileofvarious ;CRPsisrelatedtohistologicsubtypesofovariancancer ;andcanpredictthechemoresponsivenessofatumor

    ;andpatientsurviva1.

    ;Somereportshavesuggestedthatserous

    ;carcinomashadlessGSTpiactivitythannonserous

    ;tumors{andclearcellcarcinomasshowedmore ;~equentpositiveimmunostainingforGSTpithan

    ;othersubtypesoftumor[9.Incontrast.Yokoyama.

;didnotfindanydifferenceinGSTpiexpression

;betweenserousandnonseroustumors.Thecurrent

;studyiscontrasttothestudyofYokoyama,eta1.?

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